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. 2024 Apr 25;10(5):3108–3119. doi: 10.1021/acsbiomaterials.4c00305

pH- and Matrix Metalloproteinase-Responsive Multifunctional Bilayer Microneedles Platform for Treatment of Tinea Pedis

Musheng Yang †,‡,§, Lingling Pan †,‡,§, Hongmei Tian †,‡,§, Tao Zhou †,‡,§, Hui Xin †,‡, Yonglin Feng †,‡,§, Xuan Zou ‡, Ziquan Lv ‡, Yinghua Xu ∥, Xiaobao Jin †,*, Shuiqing Gui §,*, Xuemei Lu †,‡,*
PMCID: PMC11094678  PMID: 38659287

Abstract

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Persistent foot odor and itchiness are common symptoms of tinea pedis, significantly disrupting the daily life of those affected. The cuticular barrier at the site of the tinea pedis is thickened, which impedes the effective penetration of antifungal agents. Additionally, fungi can migrate from the skin surface to deeper tissues, posing challenges in the current clinical treatment for tinea pedis. To effectively treat tinea pedis, we developed a platform of bilayer gelatin methacrylate (GelMA) microneedles (MNs) loaded with salicylic acid (SA) and FK13-a1 (SA/FK13-a1@GelMA MNs). SA/FK13-a1@GelMA MNs exhibit pH- and matrix metalloproteinase (MMP)-responsive properties for efficient drug delivery. The MNs are designed to deliver salicylic acid (SA) deep into the stratum corneum, softening the cuticle and creating microchannels. This process enables the antibacterial peptide FK13-a1 to penetrate through the stratum corneum barrier, facilitating intradermal diffusion and exerting antifungal and anti-inflammatory effects. In severe cases of tinea pedis, heightened local pH levels and MMP activity further accelerate drug release. Our research demonstrates that SA/FK13-a1@GelMA MNs are highly effective against Trichophyton mentagrophytes, Trichophyton rubrum, and Candida albicans. They also reduced stratum corneum thickness, fungal burden, and inflammation in a guinea pig model of tinea pedis induced by T. mentagrophytes. Furthermore, it was discovered that SA/FK13-a1@GelMA MNs exhibit excellent biocompatibility. These findings suggest that SA/FK13-a1@GelMA MNs have significant potential for the clinical treatment of tinea pedis as well as other fungal skin disorders.

Keywords: tinea pedis, bilayer microneedles, pH- and MMP-responsive, salicylic acid, FK13-a1

1. Introduction

Tinea pedis, a chronic skin infection, results from fungal infections of the foot’s skin. It is one of the most common dermatomycoses encountered in clinical practice and is characterized by its long duration and high incidence.1,2 Typically appearing in the toe seams, soles, and heels, tinea pedis presents with interphalangeal maceration and erosion, as well as plantar damage and hyperkeratosis.3 The primary fungi responsible for tinea pedis are Trichophyton mentagrophytes, Trichophyton rubrum, and Candida albicans.4 The main treatments include imidazole and acrylamide antifungal creams and sprays.5 However, due to the stratum corneum’s unique barrier properties,6,7 these treatments are limited to delivering oil-soluble small molecules. Patients with tinea pedis often exhibit thickening of the stratum corneum, which leads to a significant reinforcement of the cuticular barrier effects that impede the penetration of antifungal agents. In addition to this thickening of the stratum corneum, fungi can infiltrate from an infected individual’s epidermis into deeper layers of skin, including fascia and bone.8 Moreover, fungi can develop resistance to chemical drugs through target-site mutations, changes in promoters, upregulation of efflux pumps, and activation of stress response pathways.9 Therefore, effective therapy for tinea pedis should enhance the permeability of antifungal agents, prevent the emergence of resistant fungi, and demonstrate exceptional antifungal and anti-inflammatory effects.

Microneedles (MNs) are known to penetrate the skin’s epidermis, creating microchannels that enable drug delivery beyond the stratum corneum barrier into the subcutaneous tissue.10 MNs offer advantages over traditional needles, including reduced pain, tissue damage, and skin inflammation.11 GelMA, a gelatin modified with methacrylate groups, exhibits double bonds introduced through modifying its amine groups.12 Its exceptional photocuring properties allow cross-linking under ultraviolet (UV) light in the presence of a photoinitiator, enhancing the mechanical strength and stratum corneum penetration capabilities of GelMA-based microneedles.13 Following the introduction of the methacrylate group, the isoelectric point (PI) of gelatin decreases leading to increased dissociation of carboxyl groups and swelling ratio in environments with pH higher than PI. Despite these modifications, GelMA retains the biological properties of gelatin and is degradable by matrix metalloproteinase (MMP),14,15 thus exhibiting pH- and MMP-responsive characteristics. The pH and MMP levels are elevated at sites of fungal infection,16,17 thereby facilitating drug release in GelMA. Salicylic acid (SA), a naturally occurring active ingredient, facilitates cutin release and enhances penetration of other drugs into deeper layers of the stratum corneum thereby improving their effectiveness.18 The integration of SA and GelMA MNs offers a novel approach for improving the permeability of transdermal drug delivery systems.

Antimicrobial peptides (AMPs) are bioactive compounds that exhibit inhibitory activity against pathogenic microorganisms. In recent decades, AMPs have emerged as potent alternatives to antibiotics, addressing the urgent need for novel nonantibiotic anti-infective agents in response to the escalating challenge of antibiotic resistance.19 LL-37, the only human antimicrobial peptide belonging to the Cathelicidin family, exhibits good antibacterial activity against both Gram-positive and Gram-negative bacteria. The antimicrobial peptide LL-37 readily binds to lipoteichoic acid or lipopolysaccharide on the bacterial cell wall, inducing disruption of the cell wall and subsequent release of intracellular components, ultimately leading to bacterial demise.20 In addition to its potent antibacterial activity, LL-37 also demonstrates antifungal efficacy against fungi. Specifically, the antimicrobial peptide LL-37 primarily exerts its fungicidal effect against C. albicans by disrupting the structural integrity of the cell wall and enhancing the permeability of the cell membrane, thereby facilitating the efflux of intracellular components.21 Furthermore, LL-37 possesses potent immunomodulatory functions that enable it to exhibit significant anti-inflammatory activity.22 FK13-a1 is a short α-spiral antimicrobial peptide derived from LL-37, consisting of 13 amino acids. Similar to LL-37, FK13-a1 exhibits potent antibacterial activity while retaining its anti-inflammatory properties.23

In this study, we propose pH- and MMP-responsive multifunctional bilayer SA/FK13-a1@GelMA MNs composed of GelMA, SA, and FK13-a1 (Figure 1). When SA/FK13-a1@GelMA MNs are applied to tinea pedis, the lower tip of the needle penetrates the epidermis to release SA, softening the stratum corneum and enhancing drug diffusion in the skin. This action facilitates the penetration of FK13-a1 into the skin, boosting its antifungal and anti-inflammatory effects. In severe cases of tinea pedis, heightened local pH levels and MMP activity further accelerate drug release. Compared with conventional methods, this study can achieve responsive release and higher transdermal efficiency of FK13-a1 in tinea pedis, thereby enabling controlled release of FK13-a1 and augmenting its antifungal and anti-inflammatory effects. Our designed SA/FK13-a1@GelMA MNs exhibit significant potential for the treatment of tinea pedis.

Figure 1.

Figure 1

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Schematic representation of SA/FK13-a1@GelMA MNs application in treating tinea pedis.

2. Materials and Methods

2.1. Materials

The FK13-a1 (WKRIVRRIKRWLR-NH2) and FITC-FK13-a1 (FK13-a1 labeled by fluorescein isothiocyanate, FITC) were obtained from GL Biochem (Shanghai) Ltd. (Shanghai, China). Terbinafine hydrochloride (TBNF HCl), salicylic acid (SA), photoinitiator (Irgacure 2959), methacrylic anhydride, and chloramphenicol were sourced from MACKLIN (Shanghai, China), while type B gelatin came from Shanghai Yuanye Bio-Technology Ltd. (Shanghai, China). Taipan Blue, nitric oxide (NO) detection kit, periodic acid-schiff (PAS) stain kit, and bicinchoninic acid (BCA) protein assay kit were acquired from Beyotime Biotechnology (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and interleukin-10 (IL-10) were purchased from Jiangsu Meimian Industrial Ltd. (Jiangsu, China). Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), glutathione (GSH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CRE) assay kits, along with the hematoxylin and eosin (H&E) stain kit, were procured from Nanjing Jianchen Bioengineering Institute (Nanjing, China). Cell Counting Kit-8 (CCK-8) from Saint-Bio (Shanghai, China). Cycloheximide was obtained from SIGMA, and sisomicin sulfate from MedChemExpress (Shanghai, China). Terbinafine hydrochloride Cream (containing 1% terbinafine hydrochloride) from Wuhan Nuoan Pharmacy Ltd. (Wuhan, China).

2.2. Synthesis of GelMA

Dissolve 5 g of type B gelatin in 50 mL of phosphate buffered saline (PBS) at 300 rpm and 50 °C. Following dissolution, methacrylic anhydride was added dropwise at a rate of 0.2 mL/min, adjusting the pH between 7.0 and 7.4 with 4 mol/L NaOH as necessary. Continue the reaction was continued at 300 rpm and 50 °C for 4 h. Then, 50 mL of PBS was added and the mixture stirred for 10 min. Dialyze the resultant mixture for 3 days using a dialysis bag with a molecular weight cutoff of 8000–14,000, followed by lyophilization to obtain white spongy GelMA.

2.3. Preparation of SA/FK13-a1@GelMA MNs

Initially, we prepared two solutions. Solution A consisted of 50 mg of photoinitiator (irgacure 2959) and 3 g of GelMA dissolved in 10 mL of 10% SA solution and mixed at 50 °C. For solution B, 50 mg of irgacure 2959 and 3 g of GelMA were dissolved in 10 mL of pure water at 50 °C and cooled to room temperature, and then 10 mg of FK13-a1 was added and thoroughly mixed. To fabricate the microneedles, 50 μL of solution A was added to a poly(dimethylsiloxane) (PDMS) microneedle mold, vacuum treated for 5 min to remove bubbles and excess solution A, dried at 50 °C for 30 min, and cross-linked with 365 nm violet light for 40 s. Then, 200 μL of solution B was added to the molds, similarly vacuum treated, and dried at 37 °C for 8 h, followed by cross-linking with 365 nm violet light for 40 s. The molds were then refrigerated and dried at 4 °C. Finally, the sample was demolded to obtain bilayer SA/FK13-a1@GelMA MNs.

2.4. Characterization

Fourier transform infrared (FTIR) spectra were collected using an FTIR Spectrometer (Nicolet iS20, Thermo Fisher Scientific, Massachusetts). 1H nuclear magnetic resonance spectra were obtained by using 1H nuclear magnetic resonance spectroscopy (AVANCE NEO 400 M, Bruker, Karlsruhe, Germany). The surface morphology of the prepared SA/FK13-a1@GelMA MNs was examined using scanning electron microscopy (GeminiSEM 300, zeiss, Oberkochen, Germany). The bilayer structure of the MNs was visualized using fluorescence microscopy (Axio Vert.A1, zeiss, Oberkochen, Germany) after loading with rhodamine B and FITC-FK13-a1. The mechanical strength of the SA/FK13-a1@GelMA MNs was evaluated using a universal testing machine (5982, INSTRON, Massachusetts).

2.5. Experiments Involving Skin Puncture

For skin puncture experiments, the SA/FK13-a1@GelMA MNs were first sterilized under a UV lamp for 15 min. The dorsal skin of the mice was depilated and disinfected with a 75% alcohol solution. During the skin penetration test, continuous pressure was applied to the MNs by using an index finger for 1 min to ensure penetration into the stretched mouse skin tissue. The microneedle patch was then gently removed, and trypan blue dye was applied to the punctured skin for 5 min to stain the area. Excess dye was removed, and digital images of the puncture site were taken to quantify and record the number of stained holes.

2.6. Swelling Degradation Under Different pH Levels and Gelatinase Concentrations

The SA/FK13-a1@GelMA MNs were submerged in PBS solutions of varying pH levels and gelatinase concentrations and incubated at 37 °C for predetermined time intervals of 15 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 24, 48 h, and finally up to a maximum duration of 72 h. Before immersion, the initial weight (Wi) of the SA/FK13-a1@GelMA MNs was measured. At each designated time point, the weight (WF) of the SA/FK13-a1@GelMA MNs was recorded after removing any residual surface water using paper towels.

2.6.

2.7. Drug Release Under Varying pH Levels or Gelatinase Concentrations

The SA/FITC-FK13-a1@GelMA MNs were immersed in 10 mL of solutions with different pH levels or gelatinase concentrations, and 100 μL of the leachate was collected at 37 °C at specific time intervals (15 min, 30 min, 1, 2, 4, 6, 8, 10, and 12 h). After each collection, 100 μL of stock solution was immediately added to replenish the lost volume. Standard curves for SA and FITC-FK13-a1 were established, and the absorbance of the leachate was measured at wavelengths of 304 and 495 nm to analyze in vitro release kinetics, using three parallel samples for each condition.

2.8. Antifungal Ratio Test

T. mentagrophytes (NBRC6202), T. rubrum (ATCC MYA-3638), and C. albicans (ATCC 10231), at a concentration of 1 × 106 CFU/mL, were inoculated into a 6-well plate with 6 mL evenly distributed among the wells. PBS, GelMA MNs, SA@GelMA MNs, FK13-a1@GelMA MNs, SA/FK13-a1@GelMA MNs, and TBNF HCl were added to each well, and the cells were incubated at 37 °C for 24 h. The fungal solution was then diluted 100-fold, and 10 μL of this solution was spread uniformly onto potato dextrose agar (PDA) plates and incubated at 28 °C for 3–5 days (2 days for C. albicans). Colony forming units (CFUs) were counted to determine the antifungal ratio. This procedure was repeated three times.

2.9. Inhibition Zone Test

Each plate was prepared in 15 mL of warm PDA with T. mentagrophytes (NBRC6202), T. rubrum (ATCC MYA-3638), and C. albicans (ATCC 10231). The concentration for each of them was adjusted to 1 × 106 CFU/mL. After cooling, the plates were treated with GelMA MNs, SA@GelMA MNs, FK13-a1@GelMA MNs, SA/FK13-a1@GelMA MNs, PBS, and TBNF HCl drug-sensitive disks (MNs prepared with a diameter of 6 mm). These materials were spaced at regular intervals. This process was replicated three times. The plates were then incubated at 28 °C for 3–5 days (1 day for C. albicans), and the diameter of the inhibition zone was measured.

2.10. In Vitro Assay for Evaluating Anti-Inflammatory Activity

We seeded 100 μL of RAW264.7 cells at a concentration of 5 × 105 cells/mL into 96-well plates and divided the experiments into Normal, Model, GelMA, SA@GelMA, FK13-a1@GelMA, and SA/FK13-a1@GelMA groups. Each treatment was repeated six times. Apart from the Normal group, all groups were treated with 1 μg/mL lipopolysaccharides (LPS) for 4 h before discarding the old medium and adding 100 μL of the corresponding drug solution to each group. After 24 h, cell supernatant from each group was collected for analysis; CCK-8 was added to assess cell viability in six wells per group. The collected supernatant was centrifuged at 3000 rpm for 10 min and then processed according to the instructions of NO detection kit and ELISA kit to measure the levels of inflammatory factors NO, TNF-α, IL-1β, IL-6, and IL-10.

2.11. Assessment of Cytotoxicity

A cell suspension containing 5 × 104 cells/mL was added to each well of a 96-well plate, with a total volume of 100 μL per well. The cells were cocultured with DMEM medium, GelMA, SA@GelMA, FK13-a1@GelMA, SA/FK13-a1@GelMA, and 15% dimethyl sulfoxide (DMSO) for either 24 or 48 h. Afterward, 10 μL of CCK-8 solution was added to each well, followed by incubation for 1–4 h. The optical density (OD) value at 450 nm was measured by using a microplate reader (varioskan flash, Thermo Fisher Scientific, Massachusetts), with six parallel replicates for each group.

2.12. Establishment and Treatment of a Guinea Pig Model of Tinea Pedis

Male SPF Hartley guinea pigs (250–300 g) were purchased from the Guangdong Medical Laboratory Animal Center. The experiments were conducted in strict accordance with the Guidelines for the Care and Use of Experimental Animals, Guangdong Pharmaceutical University (No. SYXK (Yue) 2022-0125), and they received approval from the Animal Care and Use Committee of Guangdong Pharmaceutical University, China.

T. mentagrophytes (NBRC6202) spores, at a concentration of 1 × 108 spores/mL, were used as inoculum. The epidermis of the plantar region of guinea pig hind feet was abraded with coarse sandpaper until blood spots appeared. A 0.1 mL spore suspension was then applied onto a gauze patch, which was affixed to the plantar region of the hindfoot and incubated for 7 days to establish a local infection. Afterward, the gauze was removed, and the infection was allowed to progress for another 4 weeks to induce tinea pedis. After the model was established, guinea pigs were divided into five groups, each consisting of six animals: Normal, Model, GelMA MNs, SA/FK13-a1@GelMA MNs, and TBNF HCl Cream. The guinea pigs received their respective treatments daily for 14 days, and recovery of the feet was monitored before each treatment application. Symptoms such as redness, peeling, and excessive keratinization of the foot skin were recorded. Following the 14-day treatment period, the guinea pigs were euthanized after cardiac blood sampling and their plantar skin was harvested.

2.13. Intensity of Fungal Infection Score

Animals were euthanized 1 day after the final administration. The hind feet were surgically removed and then rinsed with 0.1% benzalkonium chloride and sterile saline. The plantar skin was incised and divided into 10 blocks. These skin blocks were cultured on sabouraud dextrose agar (SDA) plates containing cycloheximide (500 mg/L), chloramphenicol (50 mg/L), and sisomicin sulfate (50 mg/L), and incubated for 10 days at 28 °C. Fungal growth on more than one skin block per foot indicated a fungus-positive foot, with the number of fungus-positive skin blocks graded from 0 to 10.

2.14. Staining of Skin Sections

Foot skin was fixed overnight in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Specimens were sectioned into 5 μm thick slices for periodic acid-schiff (PAS) and H&E staining.

2.15. Detection of Biomarkers of Oxidative Stress

A 10% skin tissue homogenate was prepared for MDA, SOD, CAT, and GSH detection. The activities or levels of these biomarkers were meticulously measured according to the instructions provided in the respective detection kit manuals.

2.16. ELISA

A 10% plantar skin homogenate was prepared, and the levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 were quantified using ELISA kits, as per the provided instructions.

2.17. Reassessment of Hepatic and Renal Functionality

Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CRE) in the guinea pigs were determined using appropriate assay kits.

2.18. Statistical Analysis

Data analysis was conducted using GraphPad Prism 9 software, with results presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used for comparisons between groups, with statistical significance set at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

3. Results and Discussion

3.1. Synthesis and Characterization of GelMA

GelMA’s outstanding mechanical properties and biocompatibility have made it a popular material in the fabrication of MNs.24Figure S1A shows the GelMA solution before and after exposure to a 365 nm UV lamp, indicating that GelMA, prepared by this method, successfully cross-links into a hydrogel. This observation confirms the successful preparation of GelMA. Fourier transform infrared (FTIR) Spectroscopy analysis, as shown in Figure S1B, reveals the combination of amino and methacrylate groups on gelatin at 3396, 1647, 1529, and 1245 cm–1, thus verifying GelMA’s successful synthesis. The 1H nuclear magnetic resonance (NMR) spectrum of GelMA (Figure S1C,D) confirms the incorporation of the methacrylate group’s double bond at 5.4 and 5.7 ppm. The degree of methacrylation (DM) of GelMA, which is defined as the ratio of methacrylate groups attached to gelatin to the number of amine groups in unreacted gelatin, can be quantified by comparing the integrated intensity of the aromatic region representing the concentration of gelatin with that of the double bond region, by integrating peaks at 7.4 ppm (aromatic residues of gelatin) and 5.4 and 5.7 ppm (double bonds of methacrylate groups),25,26 resulted in a DM of 39%.

3.2. Characterization of SA/FK13-a1@GelMA MNs

We employed a template-assisted layer-by-layer deposition and solidification technique to fabricate bilayer microneedles.27,28 As shown in Figure 2A–C, fluorescence microscopy revealed bilayer MNs consisting of a SA layer (indicated in red) and a FK13-a1 layer (shown in green), confirming the bilayer structure of the SA/FK13-a1@GelMA MNs. Scanning electron microscopy (SEM) results, presented in Figure S2, displayed the conical structure of the microneedle array, characterized by a sharp tip, a height of 600 μm, and a base diameter of 300 μm. The mechanical strength of the MNs is crucial for effective skin penetration. To evaluate this, we determined the maximum compressive force the SA/FK13-a1@GelMA MNs could withstand. As depicted in Figure 2D, each needle could endure over 0.3 N of force at a height of 600 μm, sufficient for skin penetration. Additionally, we assessed the skin infiltration ability of SA/FK13-a1@GelMA MNs by using a mouse skin model. The results, shown in Figure 2E, indicate successful infiltration of mouse skin by SA/FK13-a1@GelMA MNs.

Figure 2.

Figure 2

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Characterization of SA/FK13-a1@GelMA MNs. (A–C) Fluorescence microscopy image of bilayer MNs loaded with rhodamine B and FITC-FK13-a1. (D) Single-needle tip force–displacement curve of SA/FK13-a1@GelMA MNs. (E) After inserting the SA/FK13-a1@GelMA MNs into the skin of the mouse, images of the pinholes were captured using a camera.

3.3. In Vitro Swelling Degradation and Drug Release of SA/FK13-a1@GelMA MNs

The PI of type B gelatin is approximately 5.0, and when it reacts with methacrylate anhydride, the amino and methacrylate groups combine, leading to a lower PI for GelMA compared to that of type B gelatin. Consequently, when the pH of the GelMA solution exceeds its PI, the carboxyl group dissociates, resulting in a heightened negative charge. GelMA’s swelling ratio may increase correspondingly based on the principle of charge repulsion. As shown in Figure 3A, the swelling ratio of SA/FK13-a1@GelMA MNs progressively increases until it reaches its peak at 0.5 h and then stabilizes thereafter. The swelling ratio of MNs also increases with an increase in pH within the range of pH 5.5–7.4. GelMA includes target sequences for matrix metalloproteinase (MMP),15 which can be degraded by MMP, including gelatinase. The results depicted in Figure 3B demonstrate that the swelling ratio of SA/FK13-a1@GelMA MNs exhibits an upward trend with increasing gelatinase concentration, reaching its maximum at 0.25 h, followed by subsequent degradation. Moreover, it is observed that the degradation ratio also escalated with higher gelatinase concentrations.

Figure 3.

Figure 3

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In vitro swelling degradation and drug release of SA/FK13-a1@GelMA MNs. (A, B) Characterization of SA/FK13-a1@GelMA MNs swelling and degradation in different pH solutions. (C, F) Cumulative release ratio of SA/FK13-a1@GelMA MNs in solutions with different gelatinase concentrations. All data are represented as mean ± standard deviation (n = 3).

The active substances are released from GelMA through scaffold expansion, natural degradation, and enzymatic degradation. By utilizing the differential swelling and degradation rates of the MNs’ upper and lower layers, SA/FK13-a1@GelMA MNs release SA faster than FK13-a1. As shown in Figure 3C–F, SA achieves its peak cumulative release ratio faster than that of FK13-a1. The cumulative release ratio of SA/FK13-a1@GelMA MNs depends on both pH and gelatinase concentration. Notably, the drug cumulative release ratio of SA/FK13-a1@GelMA MNs in gelatinase solutions was significantly higher. It demonstrated faster attaining its peak cumulative release ratio than in pH solutions.

Healthy skin typically has an acidic pH ranging between 4 and 6. However, the pH of skin tissue affected by chronic injury or infection rises to 7–9.29 The association between MMP and many inflammatory diseases is well-established. Notably, the expression of MMP significantly upregulates during inflammation, with gelatinase being one prominent member of this enzyme family.30−33 Given that tinea pedis is a chronic condition often accompanied by inflammation, the affected area typically exhibits elevated pH and MMP content compared to that of normal skin. Thus, SA/FK13-a1@GelMA MNs can trigger a dual pH- and MMP-response in tinea pedis lesions, enabling faster drug release from the GelMA MNs and promoting rapid fungal eradication during severe episodes of tinea pedis.

3.4. Antifungal Activity of SA/FK13-a1@GelMA MNs In Vitro

Tinea pedis, a chronic fungal infection affecting foot skin, is caused by pathogenic agents such as T. mentagrophytes, T. rubrum, and C. albicans.34,35 To assess the antifungal efficacy of SA/FK13-a1@GelMA MNs, we conducted antifungal ratio and inhibition zone tests. As depicted in Figure 4A,B, initial assessments of GelMA MNs loaded with FK13-a1 showed significant antifungal activity against T. mentagrophytes, T. rubrum, and C. albicans, achieving an antifungal ratio close to 100%. The inhibition zone experiment further confirmed that GelMA MNs containing FK13-a1 had potent antifungal effects against T. mentagrophytes and T. rubrum compared to C. albicans (Figure 4C,D). These results highlight the exceptional antifungal properties of SA/FK13-a1@GelMA MNs against these pathogens.

Figure 4.

Figure 4

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Antifungal activity of SA/FK13-a1@GelMA MNs in vitro. (A, B) The antifungal ratio of SA/FK13-a1@GelMA MNs. The inhibitory zone of SA/FK13-a1@GelMA MNs. All data are represented as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3.5. Anti-inflammatory Activity of SA/FK13-a1@GelMA MNs In Vitro

Inflammation is a critical defense mechanism against harmful pathogens, but uncontrolled inflammation can lead to local and systemic complications, posing significant risks to the host.36,37 Therefore, managing the inflammatory response is vital in treating tinea pedis. In our study, we induced RAW264.7 cells to differentiate into M1 type using lipopolysaccharide (LPS), leading to the production of high amounts of pro-inflammatory factors such as NO, TNF-α, IL-6, IL-1β, and others that are crucial for resisting pathogen invasion and causing cell damage.38,39 We investigated the anti-inflammatory properties of SA/FK13-a1@GelMA MNs by treating M1 RAW264.7 cells with different samples. The Cell Counting Kit-8 (CCK-8) assay showed that SA/FK13-a1@GelMA significantly improved the viability of RAW264.7 cells compared to the Model group (Figure 5A), likely due to the anti-inflammatory effects of FK13-a1. Our results from NO kit detection and enzyme-linked immunosorbent assay (ELISA) demonstrated a marked decrease in the expression of NO, TNF-α, IL-6, and IL-1β and an increase in IL-10 expression in RAW264.7 cells treated with FK13-a1-loaded GelMA compared to the Model group (Figure 5B–F). These findings suggest that SA/FK13-a1@GelMA MNs possess anti-inflammatory properties.

Figure 5.

Figure 5

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Anti-inflammatory activity of SA/FK13-a1@GelMA MNs in vitro. (A) Evaluation of RAW264.7 cell viability (n = 6). (B) Determination of NO content (n = 3). (C–F) Quantification of TNF-α, IL-1β, IL-6, and IL-10 levels using ELISA (n = 4). All data are represented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3.6. Cytotoxicity of SA/FK13-a1@GelMA MNs In Vitro

Cytocompatibility is a critical factor for the clinical use of hydrogels in direct tissue contact.40 We assessed the cytotoxicity of SA/FK13-a1@GelMA MNs against NIH-3T3, HaCaT, and L929 cells using the CCK-8 assay. The cells and samples were cocultured for 24 and 48 h, followed by the addition of CCK-8 reagent. The results, presented in Figure 6A–F, revealed that apart from the notable cytotoxicity in the 15% DMSO group, the cell viability in the GelMA, SA@GelMA, FK13-a1@GelMA, and SA/FK13-a1@GelMA groups was comparable to the Normal group. These findings indicate that all materials used in the SA/FK13-a1@GelMA MNs are highly cytocompatible.

Figure 6.

Figure 6

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Cytotoxicity of SA/FK13-a1@GelMA MNs in vitro. (A–F) Cytotoxicity assay against NIH-3T3, HaCaT, and L929 cells (n = 6). All data are represented as mean ± standard deviation.

3.7. Representative Images of Tinea Pedis During Modeling and Pretherapy and Post-Treatment

The Hartley guinea pig tinea pedis model induced by T. mentagrophytes spores is a well-established and widely utilized model due to its resemblance in clinical symptoms and histopathological nonspontaneous healing to human hyperkeratosis tinea pedis.41 These analogous symptoms include erythema, hyperkeratosis, and fissuring. Figure S3 depicts a representative image of the tinea pedis modeling process in guinea pigs. By day 35, symptoms such as foot skin erythema, hyperkeratinization, and cracking were evident.

Figure 7 shows the recovery of tinea pedis after 14 days of treatment. Significant improvements in symptoms were observed in both the SA/FK13-a1@GMA MNs and Terbinafine hydrochloride (TBNF HCl) treatment groups with reductions in excessive keratinization and skin redness. These findings highlight the excellent therapeutic efficacy of SA/FK13-a1@GMA MNs for treating tinea pedis.

Figure 7.

Figure 7

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Representative images of tinea pedis treated with different drugs on days 0 and 14.

3.8. Antifungal Efficacy and Oxidative Stress Level In Vivo

Fungal load outcomes are presented in Figure 8A,B. The intensity of fungal infection score for the Model group was 7.67 ± 0.577, while the SA/FK13-a1@GelMA MNs group score was considerably lower, at 1.66 ± 0.577. One-way ANOVA analysis showed no significant difference between the SA/FK13-a1@GelMA MNs and TBNF HCl groups, indicating similar antifungal effectiveness.

Figure 8.

Figure 8

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Antifungal efficacy and oxidative stress level in vivo. (A, B) Intensity of fungal infection score. (C, F) Local oxidative stress detection. All data are represented as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Key oxidative stress biomarkers, such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH), have been extensively investigated. Recent research suggests that tinea pedis and local fungal infections can induce oxidative stress in the affected area.42 Therefore, assessing local oxidative stress levels post-treatment can be a valuable therapeutic indicator for fungal infections.43Figure 8C–F illustrates the impact of various treatments on oxidative stress levels. After 14 days of treatment, the CAT, GSH MDA, and SOD levels in the plantar skin of guinea pigs treated with SA/FK13-a1@GelMA MNs closely resembled those of the TBNF HCl group, in contrast to the significant increases observed in the Model group. These findings indicate that tinea pedis in guinea pigs, effectively treated with SA/FK13-a1@GelMA MNs for 14 days, leads to normalizing local oxidative stress levels, thereby indicating its significant efficacy in treating tinea pedis.

3.9. Histological Features of Skin Sections

As shown in Figure 9A,B, histological analysis reveals notable thickening of the stratum corneum and slight damage with irregular hyperplasia in the stratum spinosum in both the Model and GelMA MNs groups. These groups also exhibit inflammatory cell infiltration in the dermis, along with fungal spores present in both the stratum corneum and dermis. In contrast, the SA/FK13-a1@GelMA MNs and TBNF HCl groups displayed characteristics akin to those of the Normal group, with no signs of inflammatory cell infiltration and minimal spores in the dermis. Notably, SA/FK13-a1@GelMA MNs showed a therapeutic effect on tinea pedis comparable to that of TBNF HCl.

Figure 9.

Figure 9

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Histological features of skin sections. (A) H&E staining used for general morphology assessment. Blue arrows indicate the thickness of the stratum corneum. (B) Periodic acid-schiff (PAS) staining employed to evaluate specific fungal components. The red arrow points to fungi.

3.10. Anti-Inflammatory Activity of SA/FK13-a1@GelMA MNs In Vivo

The chronic nature of tinea pedis is caused by a fungal infection, with inflammation being the prominent clinical manifestation.34,35 Therefore, anti-inflammatory treatment has emerged as a pivotal therapeutic indicator for tinea pedis. Key inflammatory factors mediating skin damage in tinea pedis include TNF-α, IL-1β, IL-6, and IL-10. To evaluate the anti-inflammatory impact of SA/FK13-a1@GelMA MNs on tinea pedis, we quantified the levels of inflammatory markers TNF-α, IL-1β, IL-6, and IL-10 in skin homogenates using ELISA. Figure 10A–D illustrates a significant decrease in TNF-α, IL-1β, and IL-6 levels and an increase in IL-10 levels in guinea pigs with tinea pedis treated with SA/FK13-a1@GelMA MNs for 14 days compared to those of the Model group. These indicate that SA/FK13-a1@GelMA MNs effectively mitigate the inflammatory response.

Figure 10.

Figure 10

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Anti-inflammatory activity of SA/FK13-a1@GelMA MNs in vivo. (A–D) ELISA results of TNF-α, IL-1β, IL-6, and IL-10 contents in skin homogenates. All data are represented as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3.11. Biocompatibility Assessment of SA/FK13-a1@GelMA MNs In Vivo

Given the direct contact of SA/FK13-a1@GelMA MNs with the foot skin, it is crucial to evaluate its biocompatibility through blood biochemical analysis.44 After successfully inducing tinea pedis, the guinea pigs underwent a 14-day treatment regimen. The biocompatibility of SA/FK13-a1@GelMA MNs was assessed by measuring serum CRE, BUN, AST, and ALT levels. As shown in Figure 11A–D, the CRE, BUN, AST, and ALT levels remained within normal ranges, suggesting no nephrotoxic or hepatotoxic effects from SA/FK13-a1@GelMA MNs. These results indicate that SA/FK13-a1@GelMA MNs possess excellent biocompatibility in vivo.

Figure 11.

Figure 11

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Biocompatibility assessment of SA/FK13-a1@GelMA MNs in vivo. (A–D) Determination of CRE, BUN, AST, and ALT. All data are represented as mean ± standard deviation (n = 3).

4. Conclusions

In this study, we developed a bilayer SA/FK13-a1@GelMA MNs platform prepared from GelMA loaded with SA and FK13-a1 for the treatment of tinea pedis. Our results demonstrate that SA/FK13-a1@GelMA MNs exhibit pH- and MMP-responsive characteristics, facilitating bioresponsive drug release. Furthermore, SA/FK13-a1@GelMA MNs effectively alleviate the fungal burden, skin damage, and inflammatory response in guinea pigs with tinea pedis, while maintaining excellent biocompatibility. The therapeutic effects of SA/FK13-a1@GelMA MNs on tinea pedis can be attributed to their antifungal and anti-inflammatory properties. However, there are certain limitations associated with this study. The lack of significant superiority of SA/FK13-a1@GelMA MNs over TBNF HCl cream in the treatment of tinea pedis may be due to the comparatively lower antifungal activity of FK13-a1 compared with TBNF HCl. We propose that enhancing the antifungal potency of the loaded drugs can improve the therapeutic efficacy of the bilayer microneedle platform for tinea pedis. In conclusion, this study has successfully developed a highly penetrable bioresponsive microneedle technology platform, which has significant implications for addressing tinea pedis or other fungal skin diseases.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Nos. 32070509 and 82372161), the Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515220119), the Shenzhen Science and Technology Program (No. JCYJ20220530150412027), the Shenzhen Fund for Guangdong Provincial High level Clinical Key Specialties (No. SZGSP006), the Sanming Project of Medicine in Shenzhen (Nos. SZSM202011008 and SZSM202211016), and the Shenzhen Key Medical Discipline Construction Fund (No. SZXK066).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomaterials.4c00305.

  • Characterization images of GelMA (Figure S1); SEM image of SA/FK13-a1@GelMA MNs (Figure S2); representative images of tinea pedis during modeling (Figure S3) (PDF)

Author Contributions

X.L., S.G., and X.J. conceived the project and designed the experiments. M.Y., L.P., H.T., T.Z., H.X., Y.F., X.Z., Z.L., and Y.X. conducted the experiments and collected data. M.Y. and L.P. were responsible for the data analysis and interpretation. M.Y., L.P., X.L., S.G., and X.J. wrote the manuscript. All authors participated in result discussions and manuscript revisions.

The authors declare no competing financial interest.

Supplementary Material

ab4c00305_si_001.pdf (278.2KB, pdf)

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Supplementary Materials

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